The human papillomaviruses (HPVs) are a family of more than 80 small (approximately 8 kb) DNA viruses that infect stratified squamous epithelia causing warts. Certain high-risk HPV strains, including HPV16, HPV18, and HPV31, have been implicated as the most important etiological agents in cervical cancer [zur Hausen, Biochim. Biophys. Acta 1288:F55-78 (1996)], which is consistent with the observation that E6 and E7 genes from the high risk HPVs are potent oncogenes. Oncogenic potential of E6 and E7 may arise from binding properties to host cell proteins. For example, E6 binds to the tumor-suppressor protein p53 leading to ubiquitin-dependent degradation of the protein [Scheffher, et al., Cell 63:1129-36 (1990)], and E7 binds and promotes degradation of the tumor-suppressor retinoblastoma protein (pRB) [Dyson, et al., Science 243:934-7 (1989); Jones, et al., Genes & Dev 11:2101-11 (1997)]. While E6 and E7 have other activities, their roles in the viral life cycle are not fully elucidated.
The HPV life cycle is regulated in a differentiation-dependent manner within stratified-squamous epithelia [Jones, et al., Genes & Dev 11:2101-11 (1997)]. The virus is maintained as an episome in the basal cell layer, which is the replicating cell population in stratified epithelia. With differentiation of the host cells into keratinocytes, the virus undergoes a burst of DNA replication. Following differentiation, keratinocytes exit the cell cycle and die during the normal course of epithelial stratification. These events are normally irreversible, but HPV E7 activity is sufficient to promote an unscheduled round of DNA synthesis in differentiated keratinocytes [Cheng, et al., Genes & Dev. 9:2335-49 (1995)]. The newly synthesized viral DNA is packaged in the upper viable layers of the epithelia, and sloughed into the environment in the dead, differentiated cells. The unscheduled DNA synthesis in differentiated cells is central to the HPV viral life cycle, and the E7 gene product has been implicated as a key viral protein in this event. The E7 gene product is a 98 amino acid protein that binds a number of regulatory proteins, including pRb and proteins in the cyclin-dependent kinase inhibitory protein (KIP) family, the function of which is critical for entry into S-phase entry of the cell cycle [Morgan, Ann. Rev. Cell Dev. Biol. 13:261-291 (1997)].
How E7 promotes progression into S phase has been the subject of intense research because of the importance of this event to the viral life cycle and HPV-related cancer. E7 can overcome negative cellular growth signals including, for example, those mediated by TGF-.beta. [Pietenpol, et al., Cell 61:777-85 (1990)], loss of substrate adherence [Ruesch, et al., Virol. 250:19-29 (1998)], and serum deprivation [Pei, et al., Carcinogenesis 19:1481-6 (1998)]. This activity correlates, in part, with the ability of E7 to transform cells and bind pRb family members [Galloway, et al., Semin. Cancer Biol. 7:309-15 (1996)]. E7 binds other proteins, including, for example transcription factors such as TATA-binding proteins [Massimi, et al., Oncogene 12:2325-30 (1996)], and c-jun and c-fos family members [Antinore, et al., EMBO. J., 15:1950-60 (1996)].
Despite these binding activities, it is unclear which known function(s) of E7, if any, are key for the viral life cycle. Most notably, E7 binds pRb family members [Dyson, et al., Science 243:934-7 (1989); Ciccolini, et al., Oncogene 9:2633-8 (1994); Wu, et al., J. Virol. 67:2402-7 (1993)], p21 [Funk, et al., Genes & Dev 11:2090-100 (1997); Jones, et al., Genes & Dev 11:2101-11 (1997)], and p27 [Zerfass, et al., Oncogene 13:2323-30 (1996)], proteins that participate in the cyclin-dependent kinase phosphorylation pathway regulating cell cycle progression. The cyclin-dependent kinases regulate cell cycle progression by a variety of means [Morgan, Ann. Rev. Cell Dev. Biol. 13:261-291 (1997)], including inhibiting the ability of pRb to sequester E2F [Mulligan, et al., Trends Genet 14:223-9 (1998)], a protein that upregulates a variety of genes required for entry into S phase. E7 binding to p21 and p27, both of which inhibit CDK phosphorylation, results in a net increase in CDK2 activity. These inhibitor proteins have been implicated as key regulators of cell cycle progression that act, at least in part, via a common cyclin-dependent kinase inhibitory domain found in the amino terminus of these proteins [Polyak, et al., Cell 78:59-66 (1994); Chen, et al., Mol. Cell. Biol. 16:4673-82 (1996)]. E7 from viruses with low oncogenic potential lacks these binding activities, suggesting that interaction with one or more cellular proteins is important for neoplastic progression. Whether any of these properties are essential in the viral life cycle is unclear [Davies, et al., J. Virol., 67:2521-8 (1993); Funk, et al., Genes & Dev 11:2090-100 (1997)].
Thus there exists a need in the art to more fully determine mechanisms by which E7 is able promote viral replication and to develop methods to identify inhibitors of E7 specific activity. Inhibition of E7 can result in potent anti-viral activity and therefore, methods to identify inhibitors of E7-dependent activity are desirable.